TIMP‐1‐expressing breast tumor spheroids for the evaluation of drug penetration and efficacy

Abstract Abundance of stromal cells and extracellular matrix (ECM) is observed in breast cancer, acting as a barrier for drug penetration and presenting a key issue for developing efficient therapeutics. In this study, we aimed to develop a three‐dimensional (3D) multicellular tumor model comprising cancer and stromal cells that could effectively mimic the drug resistance properties of breast cancer. Three different types of spheroid models were designed by co‐culturing breast cancer cells (MDA‐MB‐231) with three different types of stromal cells: human adipose‐derived stromal cells (hASCs), human bone marrow stromal cells, or human dermal fibroblasts. Compared with other models, in the hASC co‐culture model, tissue inhibitor of metalloproteinases‐1 (TIMP‐1) was highly expressed and the activity of matrix metalloproteinases was decreased, resulting in a higher ECM deposition on the spheroid surfaces. This spheroid model showed less drug penetration and treatment efficacy than the other models. TIMP‐1 silencing in hASCs reduced ECM protein expression and increased drug penetration and vulnerability. A quantitative structure–activity relationship study using multiple linear regression drew linear relationships between the chemical properties of drugs and experimentally determined permeability values. Drugs that did not match the drug‐likeness rules exhibited lower permeability in the 3D tumor model. Taken together, our findings indicate that this 3D multicellular tumor model may be used as a reliable platform for efficiently screening therapeutics agents for solid tumors.

(3D) multicellular tumor model comprising cancer and stromal cells that could effectively mimic the drug resistance properties of breast cancer. Three different types of spheroid models were designed by co-culturing breast cancer cells (MDA-MB-231) with three different types of stromal cells: human adipose-derived stromal cells (hASCs), human bone marrow stromal cells, or human dermal fibroblasts. Compared with other models, in the hASC co-culture model, tissue inhibitor of metalloproteinases-1 (TIMP-1) was highly expressed and the activity of matrix metalloproteinases was decreased, resulting in a higher ECM deposition on the spheroid surfaces. This spheroid model showed less drug penetration and treatment efficacy than the other models. TIMP-1 silencing in hASCs reduced ECM protein expression and increased drug penetration and vulnerability. A quantitative structure-activity relationship study using multiple linear regression drew linear relationships between the chemical properties of drugs and experimentally determined permeability values. Drugs that did not match the drug-likeness rules exhibited lower permeability in the 3D tumor model. Taken together, our findings indicate that this 3D multicellular tumor model may be used as a reliable platform for efficiently screening therapeutics agents for solid tumors.

K E Y W O R D S
3D multicellular tumor spheroid, adipose-derived stromal cells, Collagen type-1, extracellular matrix, multiple linear regression, tissue inhibitor of metalloproteinases-1

| INTRODUCTION
Breast cancer is one of the leading causes of cancer-related deaths in women worldwide, and its incidence is increasing in all industrialized countries. 1 It is a representative solid tumor, and the breast cancer In Yeong Bae and Wooshik Choi contributed equally to this work. tumor microenvironment (TME) consists of various cell types including tumor and stromal cells, surrounding extracellular matrix (ECM), and signaling molecules. The TME has recently emerged as a critical regulator of tumor progression and drug efficacy. 2 It produces various cytokines and chemokines that regulate the composition and organization of ECM proteins. The ECM serves as a physical barrier, hindering drug entry into and diffusion throughout the tumor, thereby contributing to drug resistance. 3,4 In addition, interactions between cancer cells and stromal cells as well as various growth factors and ECM components in the TME can drastically affect the apoptotic sensitivity of cancer cells and their response to chemotherapeutic drugs. [5][6][7] In recent years, interactions between stromal cells and cancer cells have been increasingly acknowledged to be involved in tumor development and progression. Emerging evidence indicates that stromal cells in the TME play important roles in ECM production. 8 In breast cancer, tumor stromal cells such as cancer-associated fibroblasts (CAFs) mainly contribute to abnormal ECM deposition in response to several paracrine factors such as reactive oxygen species (ROS), transforming growth factor-beta (TGF-β), and platelet-derived growth factor (PDGF). 9 In addition, the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is a critical factor for stromal remodeling of the ECM. 10 Many investigations have shown that among various isoforms of TIMPs, the expression of TIMP-1 correlates with the progression and severity of several types of cancers, including colon cancer, lung carcinoma, gastric cancer, melanoma, and breast cancer. [11][12][13][14][15] As a specific hallmark of human cancer, TIMP-1 plays multiple roles related to the TME and drug resistance. Independent of its inhibitory effect on MMPs, TIMP-1 acts as a growth factor that promotes cell proliferation and tumorigenesis in various cell types, including breast cancer cells. 11,16,17 It may also function as a suppressor of apoptosis in several tumors. 18,19 Despite its diverse roles in breast cancer, it remains unclear whether TIMP-1-dependent ECM regulation affects the penetration or movement of drugs in cancer tissues.
Therefore, there is an urgent need to develop reliable platforms for screening effective therapeutic agents for breast cancer. Traditionally, the majority of cell-based in vitro assays used to develop anticancer drugs in the pre-clinical stage are facilitated using twodimensional (2D) cultures of cancer cells. However, this does not reflect the physiologic characteristics of three-dimensional (3D) tumors in vivo. This has resulted in poor prediction of drug efficacy in vivo, leading to failures in clinical trials with huge financial losses. 20 Thus, 3D culture models have recently attracted much attention as promising biotechnologies for more accurate drug efficacy screening. In contrast to 2D cell culture models, 3D tumor spheroid models recapitulate the physiologic features of human cancers in vivo in terms of heterogeneity, gene expression, signal pathways, cell-cell interactions, production/deposition of ECM, drug resistance and penetration, and 3D structure. [21][22][23][24] In addition, spheroid models are easy to control and are suitable for highthroughput drug screening. 25,26 Recent technological advances have allowed for the development of several 3D tumor models that are utilized to understand cancer progression and inhibition. [27][28][29] Specifically, 3D in vitro models that simulate the interaction between cancer cells and stromal cells have been designed for accurate evaluation of drug efficacy and penetration capacity. 30,31 However, few studies have focused on the diversity of stromal cell types used to generate 3D multicellular spheroids. To date, most research on 3D tumor models simulating the interaction between cancer cells and stromal cells has focused on using fibroblasts as a stromal component. [32][33][34][35] There remains a need for various types of 3D models that can be produced by incorporating other types of stromal cells to better understand anticancer drug resistance in the TME.
One of the important aspects of overcoming tumor drug resistance is understanding the physicochemical properties of therapeutic agents. Thus, researchers developed the notion of drug likeness, a rule that predicts whether a compound will possess the properties of a prototype drug in vivo based on its chemical properties during the early stages of drug development. Several considerations have been made to evaluate drug likeness. Analysis of the structures of drug candidates, pioneered by Lipinski with his Rule of Five (Ro5), has so far been a useful guide in achieving successful drug development. 36 Modified measurements for drug-likeness such as Veber's rule, 37 Ghose filter, 38 and quantitative estimate of drug-likeness (QED) 39 have been proposed; however, limitations still exist in predicting the actual bioavailability of drugs. 40 Frequently used in vitro experiments such as parallel artificial membrane permeability assay (PAMPA), or Caco-2 cell-based assay, could not properly recapitulate drug resistance in solid tumors, since the properties of constituent membranes are significantly different. 41,42 To the best of our knowledge, there are few in vitro models used for evaluating drug permeability and resistance in solid tumors. 43 In this study, we aimed to develop 3D multicellular tumor spheroid models comprising cancer cells and human adipose-derived stromal cells (hASCs) that could accurately evaluate drug efficacy and resistance. We also investigated and compared the morphological characteristics, as well as physical and biochemical properties of the models in relation to drug responses. To investigate the morphologic characteristics of 3D multicellular tumor spheroids, several shape parameters, such as diameter, roundness, and sphericity, were analyzed from phase-contrast images. As shown in Figure 1b, all spheroids with different stromal cells were uniformly rounded and spherical, with diameters ranging from 500 to 600 μm. These data indicate that all spheroids were consistently and uniformly generated as 3D well-defined geometrical shapes.
Next, we investigated the local distribution of breast cancer cells and stromal cells in each multicellular spheroid. Stromal and breast cancer cells were stained with green and red fluorescent dyes, respectively, and were cultured to form multicellular spheroids. In hASC-co-cultured tumor spheroids, breast cancer cells were mainly positioned in the interior of the spheroids, while most of the hASCs were distributed on the surface of the spheroids. In contrast, in hDFco-cultured tumor spheroids, the majority of breast cancer cells were located on the surface of the spheroids. In hBMSC-co-cultured spheroids, cancer and stromal cells were evenly distributed (Figure 1c). The surface structures of multicellular spheroids were analyzed using scanning electron microscopy (SEM). In hBMSC-and hDF-co-cultured tumor spheroids, the surfaces were relatively rough and mostly composed of cells. On the other hand, the surface of hASC-co-cultured spheroids mainly consisted of ECM components, and cells were hardly found on the surface (Figure 1d). Considering that the distribution of stromal cells and ECM on the surface of tumor tissue is one of the major characteristics found in in vivo breast cancer, 44 the results suggest that hASC-co-cultured spheroids mimic the in vivo tumor environment better than the other models. spheroids generated using hASC, hBMSC, or hDF along with MDA-MB-231, respectively. 3D, three dimension; hASCs, human adipose-derived stromal cells; hBMSCs, human bone marrow stromal cells; hDFs, human dermal fibroblasts; SEM, standard error of the mean 2.2 | hASC-co-cultured tumor spheroids showed higher ECM protein expression than other models To confirm whether ECM protein expression was higher in hASC-cocultured tumor spheroids than in other models, 3D multicellular spheroids were analyzed via immunofluorescence using antibodies against Collagen type I (Col I) and fibronectin, which are two major ECM components found in breast cancer. 45 Spheroids with hASCs and MDA-MB-231 showed significantly higher expression of Col I and fibronectin than the hBMSC-or hDF-co-culture model (Figure 2a,b). When spheroids were formed with a single type of cell, Col I and fibronectin were rarely expressed ( Figure S1a-c). Western blot analysis also confirmed that Col I and fibronectin expression was higher in the hASC co-culture model than in the other models ( Figure 2c). These results indicate that ECM genes were highly expressed in spheroids wherein hASCs and breast cancer cells could interact.
2.3 | ECM overexpression in the hASC-co-cultured spheroid model is associated with the TIMP-1 upregulation ECM protein expression is regulated by a balance between protein synthesis and degradation. To understand the mechanism(s) underlying the high ECM protein expression in the hASC-co-cultured tumor spheroid model, we analyzed the factors related to ECM synthesis and degradation. RNA and conditioned media from spheroids were prepared and subjected to quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The RNA levels of Col I and fibronectin, the major ECM components of the spheroids, were not significantly different among the spheroid models ( Figure 2d). In addition, TGF-β, which is known to play a major role in ECM synthesis, showed similar RNA and protein expression levels among all spheroids ( Figure S2a To investigate the factors that affect the increased expression of TIMP-1 in the hASC co-culture model, we analyzed the TIMP-1 expression in stromal cells under various conditions, including culturing as a monolayer, 3D spheroid culture, and co-culturing with breast cancer cells. In monolayer culture, stromal cells showed a higher TIMP-1 protein expression than breast cancer cells. There were no significant differences among stromal cells (Figure 2h). When hASCs were cultured in the 3D spherical configuration, TIMP-1 expression was increased by approximately fourfold compared to that in the monolayer culture; however, no significant changes were observed in other stromal cells (Figure 2h). The effect of co-culturing with stromal and breast cancer cells on TIMP-1 expression was also investigated. When hASCs were co-cultured with breast cancer cells, TIMP-1 protein expression was increased by approximately 1.25 times compared to that when the two cells were cultured separately. In the case of hBMSCs or hDFs, co-culturing with breast cancer cells did not have a significant effect on TIMP-1 protein levels ( Figure 2h,i). The RNA level of TIMP-1 showed a similar pattern; TIMP-1 expression in hASCs was significantly increased by the 3D spheroid formation and co-culturing with breast cancer cells, as compared to that in hBMSCs and hDFs ( Figure S3a-c). Taken together, these results suggest that TIMP-1 overexpression in hASC co-cultured spheroids is involved in the regulation of MMP-1 activity and ECM expression.

| Tumor spheroids co-cultured with hASC demonstrated lower drug penetration and efficacy
We next tested whether ECM deposition on the surface of spheroids would affect the penetration of anticancer drugs and their efficacy. Three types of 3D multicellular tumor spheroids were formed and treated with 10 μM doxorubicin, epirubicin, or topotecan, which are chemotherapeutic agents for various tumors, including breast cancer, for an additional 48 h. Since these chemical compounds were reported to have intrinsic fluorescence, the distribution of drugs inside the spheroids was analyzed via fluorescence microscopy. Doxorubicin, epirubicin, and topotecan were distributed throughout the treated hBMSC-or hDF-co-cultured spheroids. In contrast, there was a significant reduction in the amounts of these anticancer drugs inside the hASC-co-cultured spheroids, as compared to those in other spheroids (Figure 3a-c). When spheroids were formed with a single-cell type, the drugs were found to penetrate all types of spheroids ( Figure S4a,b). These results suggest that drug penetration is lowered specifically in the hASC-co-cultured tumor model.  (g) Enzymatic activity of MMP-1 and MMP-9 secreted from the tumor spheroids. *p < 0.05, ***p < 0.001 (one-way ANOVA), n = 3 per group. (h) TIMP-1 protein secreted from 2D-and 3D-cultured stromal cells and breast cancer cells. **p < 0.01, ****p < 0.0001 (one-way ANOVA), n = 3 per group. (i) TIMP-1 protein secreted from 2D-co-cultured stromal cells and breast cancer cells. **p < 0.01 (one-way ANOVA), n = 3 per group. For RT-qPCR analysis, values were normalized to GAPDH. All data are presented as mean ± SEM. Col I, Col1a1, 3D-a, b, f, or m; monocellular spheroid of hASCs, hBMSCs, hDFs, or MDA-MB-231, respectively. 2D-a, b, f, or m; monolayer culture of hASCs, hBMSCs, hDFs, or MDA-MB-231, respectively. 2D, two dimension; 3D, three dimension; DAPI, 4 0 ,6diamidino-2-phenylindole; Col I, Collagen type-1; Col1a1, Collagen type-1 alpha 1; ECM, extracellular matrix; hASCs, human adiposederived stromal cells; hBMSCs, human bone marrow stromal cells; hDFs, human dermal fibroblasts; SEM, standard error of the mean; MMPs, metalloproteinases; NS, not significant; TIMP-1, tissue inhibitor of metalloproteinases-1

| TIMP-1 silencing in hASC-co-cultured tumor spheroids affects ECM protein expression and drug efficacy
To test whether TIMP-1 mainly affects the increased ECM protein expression in hASC-co-cultured spheroids, hASCs were transfected with either control siRNA or siRNA against TIMP-1, followed by the As shown in Figure 6a, the drug efficacy of 16 compounds was tested in three types of spheroids: hASC and MDA-MB-231 cocultured spheroids, and monocellular spheroids formed with either hASCs or breast cancer cells, respectively. If the drug efficacy in multicellular spheroids was lower than the average efficacy in hASC and MDA-MB-231 monocellular spheroids, it might be recognized that drug penetration in multicellular spheroids was disturbed.
Therefore, we defined the permeability value (PV) of each drug as the difference in efficacy in multicellular and monocellular spheroids, as determined by Equation (4). We further investigated whether PV would correlate with canonical indicators of drug permeability, such as the PAMPA, Caco-2 permeability assay, and drug-likeness rules ( Figure 6a). As shown in Figure 6b, drugs with logP PAMPA less than À6.14, which is reported to have a low permeability, 48 had significantly lower PVs than drugs with logP PAMPA values over À6.14. In addition, drugs that were impermeable to Caco-2 monolayer cells tended to show lower PVs in the tumor model compared to the permeable drugs (Figure 6c). Multiple regression is a widely accepted statistical technique that utilizes several explanatory variables (independent variables) to predict the outcome of a response variable. Table S4 shows the values of the chemical properties of the drugs (independent variables) and their corresponding experimental PVs (output variable). We assumed that there were no instrumental variables or nonlinear terms, We also applied fivefold cross-validation for robustness. Normally, more than 30 data sets are considered sufficient for Gaussian analysis, and linear regression is performed well under the Gaussian assumption. Forward stepwise MLR analysis revealed nine descriptors used to establish the training set, and the following equation was obtained: where c PV is the predicted PV, K is the molecular weight (MW), L is

| DISCUSSION
In this study, we demonstrated that spheroids co-cultured with breast In breast cancer in vivo, the TME consists of ECM and stromal cells, such as fibroblasts, mesenchymal stromal cells (MSCs), immune cells, and vascular endothelial cells. 49 Stromal tissue in the TME reportedly plays an important role in cancer development and progression. Stromal tissues in TME have been reported to play an important role in cancer development and progression. Therefore, tumor-stromal ratio (TSR), a proportion of the stromal region inside the TME, has been identified as a promising parameter for cancer prognostication. 50 Clinically, patients with more than 50% TSR are classified as stroma rich, whereas those with less than 50% TSR classified as stroma poor: the two groups are significantly different in survival and prognosis. 51 53 or S100 calcium-binding protein A7 (S100A7), a small calcium-binding protein. 54  ( Figures S4a and 2h). These data imply that the agents derived from   Table S3. MLR, multiple linear regression; PVs, permeability values; QSAR, quantitative structure-activity relationship signaling molecules such as Smad2 and Smad3. Activated Smad protein complexes can translocate to the nucleus and act as transcription factors for various ECM genes. 57 However, TGF-β seemed to play a minor role in our case, since the RNA and protein levels of TGF-β were not significantly different among various stromal cells and breast cancer cells (Figure S2a,b). The factors that contribute to ECM synthesis in breast cancer need to be elucidated. It might be possible to inhibit ECM deposition in solid tumors more efficiently by co-regulating the ECM-promoting factor and TIMP-1, which inhibits ECM degradation. This may allow for increased penetration and efficacy of anticancer drugs inside the in vivo tumor.
TIMP-1 is secreted by various cell types in the TME. It is important to note that stromal cells such as CAFs are the primary source of TIMP-1 in solid tumors. 58 We found that 3D structure formation and co-culturing with breast cancer cells could promote TIMP-1 protein secretion in hASCs; however, the underlying mechanism by which stromal cells produce TIMP-1 has not yet been understood. One possibility is that TIMP-1 may be secreted when these cells are exposed to hypoxic conditions. It was previously reported that when cells encounter a hypoxic environment, hypoxia-inducible factor 1 alpha  (Table S1). This is in line with previous studies that indicate that 3D models are better mimics of in vivo situations; Imamura et al. have reported that breast cancer spheroids in 3D culture showed higher resistance to doxorubicin compared to monolayer cells by forming dense features and inducing an antiapoptotic environment. 61 It has been previously reported that cell packing density could affect drug penetration and resistance. Tightly packed tumor cells showed impaired penetration and relative resistance to anticancer agents compared to loosely packed cells. 62 Fibroblast tumor spheroids seemed to be more densely packed (Figure 1a,b); however, higher drug deposition and efficacy were observed in fibroblast tumor spheroids compared to hASC-and hBMSC-cocultured tumor spheroids (Figure 3a

| Morphometric analysis
The morphology of spheroids was observed using a phase contrast microscope (Zeiss, Oberkochen, Germany). Images were analyzed by ImageJ software (National Institutes of Health, MD). Sphericity Index (SI) was computed from applying the squared root into the circularity, which is obtained from ImageJ. It quantitatively indicates how the shape of the sample is similar to the spherical geometry shape as in Equation (2)

| Immunofluorescence staining
Immunofluorescence was performed as previously described. 68

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data sets generated and/or analyzed during this study are available from the corresponding author on reasonable request.